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The basolateral membrane of Atlantic lobster (Homarus americanus) epithelium possesses an electroneutral Na+ /H+ antiporter that functionally resembles members of the vertebrate NHE family. Regulatory mechanisms of this antiporter in purified hepatopancreatic E-cell suspensions, produced with a centrifugal elutriation technique, were investigated. Suspensions routinely consisted of greater than 95% E-cells displaying greater than 90% viability. Intracellular pH (pHi) was monitored by loading cells with the fluorescent dye 2´,7´-bis(carboxyethyl)-5,6- carboxyfluorescein (BCECF), and placing suspensions in a spectrofluorometer. Recovery from induced acid-loading was mediated by a Na+ -dependent, dimethylamiloride-sensitive proton efflux. Antiport activation was a sigmoidal function of pHi at values below 7.0. Addition of 20 nM phorbol 12-myristate 13-acetate (PMA) to cells suspended in a lobster physiological saline (pHo = 7.4) increased pHi from 7.2 to 7.5 over a 10-min interval. Phorbol ester-induced activation of the Na+ / H+ antiporter was due to an increased affinity for internal H+ (apparent pK was shifted toward more alkaline values) at the level of an internal H+ -binding allosteric modifier site. No effect was observed when cells were exposed to 2 µM 8-Br-cAMP. Phorbol ester activation of the lobster NHE-like Na+ /H+ antiporter was inhibited by 10 nM bisindoylmaleimide I, a potent protein kinase C inhibitor. These results taken together suggest a remarkable conservation of Na+ /H+ antiport across phyla.